Mimetic insect allatostatin analogs for insect control

ABSTRACT

Novel pseudopeptide analogs of the insect allatostatin neuropeptide family which possess biological activity mimicking that of the naturally occurring neuropeptides are disclosed. By addition of a hydrophobic moiety to an active portion of the allatostatin peptides, analogs are produced which exhibit an overall amphiphilic nature and which are capable of penetrating the insect cuticle while still retaining biological activity. Furthermore, by substituting sterically hindered amino acids or aromatic acids for any or all of the first, third or fifth amino acid residues of the allatostatin C-terminal pentapeptide, analogs may be produced which are resistant to degradation by insect peptidases while still retaining biological activity. The analogs may be used for insect control by disrupting critical reproductive and/or developmental processes normally regulated by allatostatins in insects.

BACKGROUND OF THE INVENTION

[0001] 1. Field of Invention

[0002] This invention relates to mimetic pseudopeptide analogs of theallatostatin neuropeptide family, and the use of these analogs forinsect control.

[0003] 2. Description of the Prior Art

[0004] The allatostatin family of insect neuropeptides inhibit the invitro biosynthesis of juvenile hormone (JH) by the corpora allata of thecockroaches Diploptera punctata Blattella germanica (L) and Periplanetaamericana (Belles et al., 1994, Reg. Peptides, 53:237-247; and Weaver etal., 1994, Comp. Biochem. Physiol., 107(c):107-127). In these pests, JHplays a significant role in development reproductive maturity, sexpheromone production and mating. Specifically, a reduction in endogenouslevels of JH is critical to development of the adult stage from thenymph in the cockroach, whereas oocyte growth and maturation in adultfemales show a dependency on the presence of JH (Bendena et al.,Allatostatins: Diversity in structure and function of an insectneuropeptide family. In: Beckwith et al., Eds., Neuropeptides inDevelopment and Aging, pp. 53-66, Annals NY Acad. Sci. 814:53-66: 1997).

[0005] Although the allatostatins can influence a number ofphysiological processes by virtue of their ability to modulate in vitroproduction of JH, the native allatostatins have held little promise asinsect control agents. The major limitations of the allatostatins whichhave hampered their use for insect control include their inability topenetrate the insect cuticle, and their susceptibility to inactivationby peptidases in the hemolymph and gut and/or bound to tissues withinthe insect (Bendena et al., ibid).

[0006] Earlier structure-activity studies have shown that the C-terminalpentapeptide Xaa₃-Xaa₁-Phe-Gly-Xaa₂-NH₂ (wherein Xaa₁ occurs as any ofAsn, Asp, Gly, Ser or Ala, Xaa₂ occurs as Leu or Ile., and Xaa₃ occursas Tyr or Phe) is shared by all members of the Diploptera allatostatins,and represents the ‘active core’ region or minimum sequence capable ofeliciting inhibition of JH production in vitro (Hayes et al., Peptides,15:1165-1171, 1994; Pratt et all., Biochem. Biophys. Res. Commun.,163:1243-1247, 1989; and Pratt et al., Proc. Natl. Acad. Sci. USA,88:2412-2416, 1991). The side chains of active core residues Phe, Leuand Tyr proved to be the most important for activity (Hayes et al.,ibid). Recent studies have elucidated the primary catabolic cleavagesites of the allatostatins following incubation with hemolymph enzymesand with membrane peptidases in crude membrane preparations. Hemolymphenzymes primarily cleave the peptides in the N-terminal region outsideof the pentapeptide core region and therefore, do not inactivate theallatostatins (Garside et al., Peptides, 18:17-25, 1997). However,membrane preparations of brain, gut and corpora allata cleaveallatostatins at the C-terminus between residues Gly-Leu, with secondarycleavage occurring between the residue block Xaa₃-Xaa₁ (Garside et al.,Gen. Comp. Endocrinol., 108:258-270, 1997). Both of these cleavagesdisrupt the active core sequence and lead to completely inactivefragments.

[0007] The contents of each of the above-mentioned publications areincorporated by reference herein.

SUMMARY OF THE INVENTION

[0008] We have discovered novel pseudopeptide analogs of the insectallatostatin neuropeptide family which possess biological activitymimicking that of the naturally occurring neuropeptides. By addition ofa hydrophobic moiety to an active portion of the allatostatin peptides,analogs are produced which exhibit an overall amphiphilic nature andwhich are capable of penetrating the insect cuticle while stillretaining biological activity. Furthermore, by substitutingsterically-hindered amino acids or aromatic acids for any or all of thesecond, third or fifth amino acid residues of the allatostatinC-terminal pentapeptide, analogs have been produced which are resistantto degradation by insect peptidases while still retaining biologicalactivity. The analogs may be used for insect control by disruptingcritical reproductive and/or developmental processes normally regulatedby allatostatins in insects.

[0009] In accordance with this discovery, it is an-object of thisinvention to provide novel compounds having biological activitymimicking that of the naturally occurring allatostatin neuropeptides.

[0010] It is also an object of this invention-to provide compounds whichare bioactive mimics of allatostatin neuropeptides that are capable ofpenetrating the insect cuticle.

[0011] Another-object is to provide compounds which are bioactive mimicsof allatostatin neuropeptides which are resistant to enzyme degradation.

[0012] Yet another object is to provide compounds which are bioactivemimics of allatostatin neuropeptides and their use for controllinginsect populations and which may be topically applied.

[0013] Other objects and advantages of the invention will become readilyapparent from the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows the effect of time on the penetration of the9-fluoreneacetic acid pentapeptide analog of Example 1 through thecuticle of the cockroach.

[0015] FIGS. 2(a)-(c) show the structures of peptidase resistantallatostatin analogs containing sterically hindered, restrictedconformation components: a) indane ring (Aic) analog 396-1, top left; b)cyclopropyl ring (Cpa) analog 397-2, top right; and c) cyclopropyl ring(Cpa) analog with a hydrocinnamic acid (Hca) ‘cap.’ replacement-for Phe[AST(b)φ2], bottom center. Upward arrows denote cleavage sites oftissue-bound peptidases in the cockroach. The large upward arrows with across over them, indicate a peptidase cleavage site-that is blocked bythe presence of the sterically hindered components and/or presence ofthe hydrocinnamic acid cap.

[0016] FIGS. 3(a) and (b) show-the structures of peptidase resistantallatostatin analogs containing sterically hindered, restrictedconformation components: a) both an indane ring (Aic) and cyclopropylring (Cpa), and b) benzodiazepine (Bzd).

[0017]FIG. 4 shows the dose-response curve for inhibition of in vitrojuvenile hormone JH biosynthesis in corpora allata of the cockroachDiploptera punctata by allatostatin analog AST(b)φ2 in Example 2. Eachpoint for analog treatment represents the mean of 14-16 measurements +/−the standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

[0018] In the following description, the nomenclature used to define thepeptides and pseudopeptides is that specified by Schroder and Lubke[“The Peptides,” Academic Press (1965)] wherein, in accordance withconventional representation, the N-terminal appears to the left and theC-terminal to the right. Where the amino acid residue has isomericforms, it is the L-form of the amino acid that is represented hereinunless otherwise expressly indicated.

[0019] Allatostatin analogs capable of penetrating the insect cuticleare prepared by conjugating a hydrophobic moiety to a member of theallatostatin insect neuropeptide family, or a bioactive portion thereof,to render the compound amphiphilic. These-compounds are of the generalformula I:

R—X₁-Phe-Gly-X₂—NH₂  (I)

[0020] wherein X₁ is Asn, Asp, Gly, Ser, or Ala, and X₂ is either Leu orIle. The R moiety incorporates the hydrophobic functionality which iseffective to impart the amphiphilic nature to the molecule.

[0021] In a first preferred embodiment, the compound is apseudopentapeptide analog of the C-terminal allatostatin core region. Inthis embodiment, a hydrophobic carborane moiety is incorporated onto theN-terminus of the C-terminal pentapeptide, Xaa₃-X₁-Phe-Gly-X₂—NH₂ (Xaa₃being Phe or Tyr), or the C-terminal tetrapeptide, X₁-Phe-Gly-X₂—NH₂.Specifically, o-, m- or p-carborane conjugated to a short-chainalkanoyl-acyl group is conjugated to the N-terminal Tyr or Phe aminoacid of the pentapeptide, or the N-terminal X₁ of the tetrapeptide. In avariation of this embodiment, the aromatic ring of the Tyr or Phe aminoacid (Xaa₃)-may be replaced by a carborane moiety, thereby formingcarboranyl alanine of the formula:

[0022] Referring to formula I, in this first preferred embodiment R maytherefore be shown as:

Cb-(CH₂)_(n)—C(O)—X₃′  (III)

[0023] where Cb is a carborane, n is 1, 2 or 3, and X₃′ is Tyr, Phe,carboranyl alanine or a bond. When X₃ is a bond, n is preferably 2.

[0024] In a second preferred embodiment, the allatostatins or an activeC-terminal portion thereof containing at least the C-terminalpentapeptide Xaa₃-X₁-Phe-Gly-X₂—NH₂ (Xaa₃ being Phe or Tyr), aremodified, at their N-terminus by addition of a hydrophobic moiety whichmay be an aromatic amine, aromatic acid, or aliphatic fatty acid. Avariety of hydrophobic aromatic amines and acids are suitable for useherein. Preferred acids include phenyl alkanoic, alkenoic or alkynoicacids such-as 9-fluoreneacetic acid, 6-phenyl hexanoic acid or 9-phenylnonanoic-acid, while preferred amines include phenyl alkanoic, alkenoicor alkynoic amines such as 4-phenyl butyl amine. Without being limitedthereto, examples of other suitable aromatic acids include2-biphenylenecarboxylic acid, 9-anthracenecarboxylic acid,1-fluorenecarboxylic acid, and 1-pyrenebutyric acid, while othersuitable aromatic amines include 1-aminoanthracene,6-amino-3,4-benzocoumarin, 2-amino-7-bromofluorene,6-aminochrysene-3-aminofluoranthene, 9-aminophenanthrene, and1-pyrenemethylamine, and suitable aliphatic fatty acids include palmiticacid, caprylic acid, decanoic acid, lauric acid, and valeric acid.Optionally, the hydrophobic aromatic amines or acids or the aliphaticfatty acids may further include the amino acid Arg conjugated thereto.

[0025] The analog of this second embodiment may be prepared from anymember of the allatostatin family of neuropeptides. A variety of theseallatostatins have been previously described and are suitable for useherein. As mentioned, the allatostatin polypeptide to which thehydrophobic moiety is attached should include at least an activeC-terminal portion containing the C-terminal pentapeptideXaa₃-X₁-Phe-Gly-X₂—NH₂, and may encompass the entire native allatostatinpolypeptide. However, the polypeptide should not be so large as to losethe hydrophobic character introduced by the hydrophobic moiety. Thus,without being limited thereto, the size of the polypeptide (includingthe above-mentioned C-terminal pentapeptide) is usually less than orequal to about 20 amino acids, particularly less than or equal to about10 to 12 amino acids. As described in the first embodiment, the aromaticring of the Tyr or Phe amino acid (Xaa₃) may again be replaced by acarborane moiety (i.e. substituting carboranyl alanine for the Phe orTyr).

[0026] The above-mentioned hydrophobic aromatic acids or amines or thealiphatic fatty acids may be conjugated to the allatostatin peptidedirectly or through an optional spacer. Use of the spacer is preferredhowever, to minimize any steric hindrance of the active polypeptideportion of the compound by the hydrophobic moiety and inhibition ofreceptor binding. The structure of the spacer will vary with theparticular hydrophobic group. Without being limited thereto, when thehydrophobic moiety is an aromatic acid or an aliphatic fatty acid,preferred spacers may be non-polar hydrocarbons having a free aminogroup and free carboxyl group, or relatively non-polar or unchargedα-amino acids, such as Ala, Ala-Ala dimer, or Gly. When using anaromatic amine as the hydrophobic moiety, preferred spacers arehydrocarbon diacids, such as succinic acid. Other specific spacers maybe readily determined by the practitioner skilled in the art.

[0027] In summary, in accordance with the structure shown in Formula(I), the structure of R for the second embodiment may be shown as:

R₁-L_(m)-X₄—R₂—X₃—  (IV)

[0028] where R₁ is the above-mentioned hydrophobic moiety, L is thespacer, m is 0 or 1, X₄ is a bond or Arg, and X₃ is Tyr, Phe orcarboranyl alanine. The group R₂ may be a bond, or an amino acid orpolypeptide which is naturally contiguous to the C-terminal pentapeptideXaa₃-X₁-Phe-Gly-X₂—NH₂. Without being limited thereto, specific examplesof suitable R₂ moieties include: -Leu-,-Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-  Val-, -Ser-Lys-Met-,-Asp-Gly-Arg-Met-, -Asp-Arg-Leu-, -Ala-Arg-Pro-,-Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-, -Gly-Gly-Ser-Leu-,-Gly-Asp-Gly-Arg-Leu-, -Pro-Val-Asn-Ser-Gly-Arg-Ser-Ser-Gly-Ser-Arg-,-Tyr-Pro-Gln-Glu-His-Arg-, and -Pro-.

[0029] In a third embodiment for the preparation of allatostatin analogscapable of penetrating the insect cuticle, the aromatic ring of the Tyror Phe amino acid (Xaa₃) is replaced by a carborane moiety. While thismodification is the same as described in the first and secondembodiments (substituting carboranyl alanine for the Phe or Tyr), thisembodiment does not require the use of additional hydrophobic groups asdescribed in the earlier embodiments. The resultant pseudopentapeptideanalog may include an optional amino acid, Arg, conjugated to the freeamine group of the carboranyl alanine.

[0030] In summary, referring to Formula 1, in this third embodiment thestructure of R may be shown as:

R₃-carboranyl alanine-

[0031] wherein R₃ is H or Arg.

[0032] In accordance with a further embodiment, the invention alsoencompasses the preparation of allatostatin analogs which are resistantto degradation by insect peptidases while still retaining biologicalactivity. These analogs may be prepared by substituting selectedsterically hindered amino acids and/or aromatic acids for the second,third and/or fifth amino acids of the allatostatins or active C-terminalportions thereof containing the C-terminal pentapeptideXaa₃-X₁-Phe-Gly-X₂NH₂ (Xaa₃, X₁, and X₂ being as described above).

[0033] The substituted amino acids or aromatic acids incorporated intothe pentapeptide must possess sufficient bulk as to sterically hindercleavage of the analog by peptidases in the target insect, yetsubstantially retain the secondary structure and active conformation ofthe native allatostatin pentapeptide necessary for biological activity.In this embodiment, the Phe of the native C-terminal pentapeptide isreplaced with 2-amino-indane-carboxylic acid (Aic), and/or the Gly isreplaced with either cyclopropyl alanine (Cpa) or cyclobutyl alanine(Cba). Alternatively, both the Phe and Gly may be replaced with1,4-benzodiazepine (Bzd). These substitutions may be incorporated intothe complete allatostatins or into active C-terminal portions thereofwhich contain the C-terminal pentapeptide.

[0034] All of the Bzd, Cpa, and Aic containing analogs retainsignificant biological activity and increased resistance to peptidasedegradation. The Aic containing analogs in particular exhibit biologicalactivity comparable to the native allatostatin C-terminal pentapeptide.Each of the Aic, Cpa, and one of the diastereomers of the Bzd containinganalogs exhibit inhibition of JH biosynthesis in the cockroach in thephysiological (μM or lower) range. Interestingly, rather than inhibitingJH biosynthesis, the other diastereomer of the Bzd containing analogactually stimulates JH biosynthesis in the cockroach. Despite theseapparent differences, all of the analogs are effective for insectcontrol as described hereinbelow. Although these analogs exhibitresistance to degradation by hemolymph peptidases as well as membranebound peptidases at their-primary cleavage site, between Gly and Leu-NH₂in the native pentapeptide, they are still susceptible to degradation bythe membrane bound peptidases at their secondary cleavage site near theN-terminal end of the pentapeptide.

[0035] In contrast to the allatostatin pentapeptide analogsincorporating sterically hindered amino acids at the second or thirdamino acids, analogs prepared by substitution of hydrocinnamic acid(Hca) or hydroxycinnamic acid (Hhca or hydroxyphenyl propionic acid) forthe N-terminal Xaa₃ amino acid (Tyr or Phe) of the C-terminalallatostatin pentapeptide, exhibit significantly increased resistance todegradation by membrane bound peptidases near the N-terminus of themolecule. These analogs also retain significant biological activity. TheHca or Hhca replace the N terminal Tyr or Phe, capping the peptide andeffectively removing the N-terminal cleavage sites of the membrane boundpeptdases within the native pentapeptide, and also blocking theN-terminal primary cleavage sites of the hemolymph enzymes.

[0036] While the above-described Cpa, Aic, Bzd, and Hca (or Hhca)substitutions may be incorporated into the analog individually, in thepreferred embodiment, optimal resistance to insect peptidases isachieved by substitution of each of the second, third and fifth aminoacids as described. Thus, in the preferred embodiment; the analogs willincorporate both the substitution of an Hca or Hhca for Xaa₃, andsubstitution of the Phe or Gly with Aic or Cpa, respectively, orsubstituting both Phe and Gly with Aic-Cpa or with Bzd.

[0037] In review, the peptidase-resistant allatostatin analogs of thisinvention may be generally shown by the formula:

R′—X₁—X_(a)—X_(b)—X₂—NH₂  (V)

[0038] wherein X₁ and X₂ are as described above in formula I. R′ may bePhe, Tyr, a hydrocinnamyl group, a hydroxyhydrocinnamic acyl group, or apolypeptide group which may be all or a portion of an allatostatinneuropeptide which is naturally contiguous to the C terminaltetrapeptide X₁-Phe-Gly-X₂—NH₂. X_(a)-X_(b) may be any of Phe-Caa,Aic-Gly, Aic-Caa, or Bzd, wherein Caa is a cycloalkyl alanine selectedfrom cylcopropyl alanine or cyclobutyl alanine, Aic is a2-amino-indane-2-carboxyl group, and Bzd is a 1,4-benzodiazepine group.Detailed structures of peptidase resistant analogs representative ofthis embodiment are shown in FIGS. 2 and 3. The peptidase resistantanalogs of this invention are also described in Nachman et al. (1997,Active Conformation and Peptidase Resistance of ConformationallyRestricted Analogues of the Insect Allatostatin Neuropeptide Family, IN:Kawashima and Kikuyama, Advances in Comparative Endocrinology, Bologna,Italy: Monduzzi Editore, pp. 1353-1359), Garside et al. (1997,Catabolism of Insect. Neuropeptides: Allatostatin as Models forPeptidomimetic Design, IN: Kawashima and Kikuyama, Advances inComparative Endocrinology, Bologna, Italy: Monduzzi Editore, pp.169-173), and (1998, Bioorganic & Medicinal Chemistry, 6:1379-1388) thecontents of each of which are incorporated by reference herein.

[0039] Although the preparation and use of analogs capable ofpenetrating the insect cuticle and analogs resistant to peptidasedegradation may be practiced separately, it is also understood thatanalogs may be prepared which incorporate the modifications of eachembodiment. Thus in a particularly preferred embodiment, a hydrophobicmoiety is conjugated to the allatostatin-peptide or a bioactive portionthereof, while one or both of Aic or Cpa are substituted for the Phe orGly, respectively, or Bzd is substituted for both Phe and Gly, withinthe C-terminal pentapeptide core.

[0040] The analogs of this last embodiment may be shown by the formula:

R—X₁—X_(a)—X_(b)—X₂—NH₂  (VI)

[0041] wherein R, X_(l), and X₂ are as described in formula I, andX_(a)-X_(b) are as described in formula V hereinabove.

[0042] The peptides and pseudopeptide analogs of this invention may besynthesized by any suitable method, such as exclusively solid-phasetechniques, partial solid-phase techniques, fragment condensation, orclassical solution addition. The groups or amino acids of the compoundsof the invention, including the R groups, are typically joined toadjacent groups through amide linkages. The peptides may also besynthesized by recently developed recombinant DNA techniques which maybe utilized for large-scale use.

[0043] Synthesis by the use of recombinant DNA techniques, for thepurpose of this application, should be understood to include thesuitable employment of structural genes coding for the sequence asspecified hereinafter the synthetic peptides may also be obtained bytransforming a microorganism using an expression vector including apromoter or operator, or both, together with such structural genes andcausing such transformed microorganisms to express the peptide.

[0044] As stated-above, the compounds of formulas I-VI can besynthesized by methods well known to those skilled in the art of peptidesynthesis, e.g., solution phase synthesis [see Finn and Hoffman, In“Proteins,” Vol. 2, 3rd Ed., H. Neurath and R. L. Hill (eds.), AcademicPress, New York, pp. 105-253 (1976)], or solid phase synthesis [seeBarany and Merrifield, In “The Peptides,” Vol. 2, E. Gross and J.Meienhofer (eds.), Academic Press, New York, pp. 3-284 (1979)], orstepwise solid phase synthesis as reported by Merrifield [J. Am. Chem.Soc. 85: 2149-2154 (1963)], the contents of each of which areincorporated herein by reference. In the preferred embodiment, theallatostatin polypeptides and the analogs may be synthesized using thesame solid phase, techniques described by Nachman et al. (1995, Reg.Peptides. 57:359-370) or Christensen et al. (1991, Proc. Natl. Acad.Sci., USA, 88:4971-4975), the contents of each of which are incorporatedby reference herein. Cbe or 2-o-carboranylpropanoic acid may besynthesized according to previously described procedures (Radel andKahl, 1993, Amino Acids, 5:170, the contents of which are incorporatedby reference herein). The hydrophobic moieties may be incorporated intothe analogs using the techniques described in Nachman et al. for thepreparation of pyrokinin analogs (U.S. Pat. No. 5,795,857, issued Aug.18, 1998, the contents of-which are incorporated by reference herein).

[0045] The pseudopeptide analogs of formulas I-VI mimic the biologicalactivity of the naturally occurring allatostatin neuropeptides and havedemonstrated the ability to disrupt or alter physiological processesfollowing application to an insect. In a preferred embodiment, with oneexception, the pseudopeptide analogs of each of the embodiments areeffective for inhibiting juvenile hormone production in cockroaches,thereby causing infertility in adults. Furthermore, inhibition ofjuvenile hormone production in immature insects causes precocious adultdevelopment resulting in sexual dysfunction. As noted above, rather thaninhibiting juvenile hormone biosynthesis, one of the diastereomers ofthe Bzd containing analogs stimulates production of the hormone incockroaches. Application of these analogs to immature insects mayprevent or inhibit their development to adults. The compounds appear tobe particularly suited for control of insects of the order Dictyopteraand Orthoptera, especially cockroaches and locusts.

[0046] As a practical matter it is-anticipated that compositions of thepseudopeptide analogs would be prepared by formulating the compoundswith an agriculturally acceptable inert carrier, particularly a solventsuitable for topical applications. Although a variety of solvents may beused, water is preferred. The compounds may also be formulated withsolid inert carriers such as talc, clay or vermiculite, or incorporatedinto conventional controlled release microparticles or microcapsules. Inaddition, the analogs may be optionally formulated in combination withconventional insect attractants, baits, or other chemical or biologicalinsecticides.

[0047] The allatostatin analogs of this invention may be applieddirectly to the target insects (i.e., larvae, pupae and/or adults), orto the locus of the insects. Because the compounds incorporatinghydrophobic moieties will penetrate the insect cuticle, they arepreferably administered topically, such as by direct spraying on theinsect or a substrate which is likely to be contacted by the insect.Alternatively, the compounds may also be administered eithersubcutaneously, percutaneously, or orally. When they are to be ingested,they should be applied with their carrier to the insect diet. Thecompounds are administered in an amount effective to induce the desiredresponse as determined by routine testing. For example, where thedesired effect is the inhibition of juvenile hormone biosynthesis, an“effective amount” is defined to mean those quantities which will resultin a significant decrease in juvenile hormone production in a test groupas compared to an untreated control. Similarly, where the ultimateresponse is pest mortality, an “effective amount” is defined as thosequantities which will result in a significant mortality rate in a testgroup as compared to an untreated control. The actual-effective amountwill of course vary with the specific compound, the target insect andits stage of development, the application technique, the desired effect,and the duration of the effect, and may be readily determined by thepractitioner skilled in the art. When determining effective amounts, itis understood that these analogs need not be as potent as the naturalallatostatin peptide to disrupt physiological processes such as juvenilehormone biosynthesis, because their effects can be exerted over aconsiderable time, as a consequence of their resistance to peptidasedegradation. Without being limited thereto, it is envisioned that whenadministering the analogs by ingestion, effective inhibition of juvenilehormone biosynthesis may be achieved using concentrations of betweenabout 100-500 picomoles/insect. When the compounds are to be topicallyapplied, the effective amounts may be significantly higher.

[0048] It is envisioned that the compounds encompassed herein may beeffective for controlling a variety of insects. Without being limitedthereto, pests of particular interest are agronomically or commerciallyimportant insects, especially cockroaches and locusts.

[0049] The following examples are intended only to further illustratethe invention and are not intended to limit the scope of the inventionwhich is defined by the claims.

EXAMPLE 1 Materials and Methods

[0050] Cuticle Preparation

[0051] Adults of the American cockroach were obtained from colonies atour facility (CMAVE). Animals, anesthetized by submersion in H₂O for 30min, were pinned ventral side up in a wax dissecting dish flooded withwater. Lateral incisions along the margins of the abdominal sterniteswere made between segments-1-7. The epidermal layers including thecuticle and epidermal-cells were lifted up and associated tissue wascleared using forceps prior to removal. The epidermal tissue was placedcell side up on a microscope slide and the cells were scraped from thecuticle using a glass cover slip. To further clean the cuticle ofcellular debris the tissue was floated, cell side down, in a beakercontaining H₂O and subjected to sonication in a water bath at 30° C. for30 min. The cuticle strips were then washed 3× in clean water. Pieces ofcuticle, ca. 0.4 cm², composed of individual sternites withoutassociated intersegmental membranes were then dissected away fromremaining cuticle.

[0052] Incubation of Cuticle

[0053] Incubation of cuticle was accomplished using the wells of ELISAplates (Corning, 96 well Easy Wash). Prior to use the wells of theplates were blocked to minimize adsorption of analogs to the wells byfilling with a 1% gelatin in 10 mM sodium phosphate buffer,containing-150 mM NaCl (pH 7.25) (PBS) and incubating at 35° C. for 1.5h. After blocking, the plates were washed with PBS-Tween and followed bythree washes with H₂O. The wells of the plates were filled with 300 μlof H₂O and pieces of cuticle were floated, cell side down, in the wells.The 9-fluoreneacetic acid pentapeptide analog. (0.5 nmol) was applied tothe center of the cuticle pieces in a 0.5 μl drop of H₂O using aHamilton 10 μl syringe fitted with a fused silica needle (0.17 mm OD)and held in a Brinkmann micromanipulator. All applications and transferswere made using a microscope and the preparations were observed for 5min after application of the analogs to insure that drops did not slideoff the cuticle. Data from wells in which the drops slid off the cuticlewere not considered for analysis, Lids were applied to the plates-afterthe drops of water containing the analogs dried (ca. 5 min afterapplication) and the plates were placed on an orbital shaker operated at80 rpm. At this speed no water contacted the upper surface of thecuticle. One h after application of the analogs we carefully transferredthe cuticle to new wells containing 300 μl of H₂O and incubation wascontinued. Cuticle was subsequently transferred to new wells atintervals after application of the analog. After incubation, 100 pmol ofinternal standard were added to the wells. The contents of the wellswere mixed by pumping the contents in and out of a micro pipette severaltimes and then transferred to a 1.5 ml microfuge tube. The wells wereextracted two additional times by addition of 350 μl of water and theextracts combined prior to concentration to apparent dryness using aSpeedVac concentrator.

[0054] Dried samples were reconstituted in 35% acetonitrile (MeCN)(Burdick and Jackson) prior to analysis. Reversed phase liquidchromatographic analysis (RPLC) was accomplished using a LDC Biochromequaternary gradient pump and LDC Spectro Monitor 3200 UV detector set at210 nm and interfaced to a Nelson Analytical 3000 data acquisition andanalysis system. A Macrosphere 300 C18 reversed phase column (250 mm×2.1mm id, 5 μm, Alltech) was used for all separations. Solvents used forall separations were H₂O and MeCN each containing 0.1% TFA as the ionpair reagent. Samples were injected onto the column using a Rheodyne7125 injector (100 μl loop) in 35% MeCN. The column was eluted after a 5min equilibration period using a linear gradient from 35%-75% MeCN over90 min at a flow rate of-0.25 ml/min. Analysis of equimolar amounts ofanalogs indicated that the compounds had different detector responseswhen analyzed with the UV detector set at 210 nm. Therefore, all valueswere corrected to reflect the differential detector response for theanalogs. Data acquired from analyses were reduced and analyzed usingNCSS 97 software using regression analysis and T-Tests.

[0055] Radiochemical Assay

[0056] Rates of JH release were determined by the in vitro radiochemicalmethod of Feyereisen and To be (Anal. Biochem., 111:372-375, 1981) andas modified by To be and Clarke (To be and Clarke, Insect Biochem.,15:175-179, 1985) The incorporation of L[¹⁴C-S-methyl]-methionine (50μM, specific radioactivity 1.48-2.03 GBq/mmol from New England Nuclearor Amersham) into JH III at its penultimate step of biosynthesis by CAincubated in 50 μl TC 199 (GIBCO, 1.3 mM Ca²⁺, 2% Ficoll,methionine-free) was used to quantify JH release. Animals wereanaesthetized on ice prior to dissection. Corpora-allata were dissecteddirectly into non-radioactive medium (only animals with oviposited basaloocytes were used for assay). Samples were dried with nitrogen andresuspended in 10 μl 1N HCl. Radioactive medium was added andneutralized with 10 μl NaOH. Individual CA were transferred fromnon-radioactive medium to radioactive medium and were incubated for 3 h.The amount of inhibition was expressed as the percent reduction from theuntreated rate: [1−(treated rate/untreated rate)]×100%. Each valuerepresents replicate incubations of a minimum of 8 tests.

[0057] Controls

[0058] All experiments were run with appropriate controls (Dip-AST5incubated for 120 min with no membrane preparations added). If theidentical amount of AST was incubated under the same conditions withsaline alone, the size of the HPLC-detected peak (U.V. 214 nm) remainedconstant over a 120 min period. The addition of 200 μl of 30% aqueousTFA completely inactivated the enzymes in the membrane preparations andin intact CA.

Results

[0059] Analysis of samples obtained when using cockroach cuticleindicated that the analog penetrated the cuticle effectively (FIG. 1).The total amount of each of the analogs recovered over time increased ina logarithmic fashion (r=0.988, n=7/sample interval) over the testperiod. Although the amount released per hour declined significantlyduring the first six hours after application the release rate stabilizedafter six hours to about 1.5 pmol/h. The logarithmic rates of release ofthe three analogs through the cuticle of the cockroach demonstrated thatthe cuticle was acting as a slow release matrix for the analog. Theinitial rapid rates of release indicated that the cuticle at the regionof application was saturated with pseudopeptide and that the analog waspenetrating at its maximal rate. As time progressed, analog was absorbedand distributed more evenly in the cuticle surrounding the point ofapplication. Therefore, equilibrium was established throughout thecuticle so that the rate of release was reduced and declined slowly in alinear fashion.

[0060] The activity of, the 9-fluoreneacetic acid pentapeptide analog(analog 525) was 2.6×10⁻⁷ M (ED₅₀ or EC₅₀). In another assay, theactivity of the carborane containing analog, carboranyl propanoicacid-Ser-Phe-Gly-Leu-NH₂, was 6.442×10⁻¹⁰ M (ED₅₀)

EXAMPLE 2 Materials and Methods

[0061] Allatostatin Analog Synthesis

[0062] The allatostatin analogs 396-1(Ala-Arg-Pro-Tyr-Asn-Aic-Gly-Leu-NH₂, Aic=2-amino-indane-2-carboxyl-)and 397-2 (Ala-Arg-Pro-Tyr-Asn-Phe-Cpa-Leu-NH₂, Cpa=cyclopropylAla) weresynthesized as previously described Nachman et al. (1997, 1998, ibid).The pseudopeptide allatostatin analog AST(b)φ2 (Hca-Asn-Phe-Cpa-Leu-NH₂,Hca=Hydrocinnamyl- and Cpa=cyclopropylAla-) was synthesized utilizingFMOC protection chemistry on MBHA resin (0.97 meq/gm substitution;Advanced Chemtech, Louisville, Ky.). Coupling reagents used for themajority of the amino acid condensations were 1 eq. of 1,3-diisopropylcarbodiimide/1-hydroxy-7-azabenzotriazole (HOAt) mixturein-dimethylsulfoxide for 2 h according to previously describedprocedures (Nachman et al., Peptides, 18:53-57, 1997). However, forcoupling of the Cpa residue and the residue immediately following Cpa tothe peptide-resin complex the reagent used was 1 eq.[O-(7-azabenzotiazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate] (HATU)(PerSeptive Biosystems, Marlborough, Mass.)with 2 eq. N,N-diisopropylethylamine in dimethylsulfoxide for 4 h.Removal of the N-terminal FMOC group from the Cpa was accomplished with20% piperidine in dichloromethane for 1 h rather than the 30 min usedfor the other residues. The peptide was cleaved from the resin with HFaccording to previously described conditions (Nachman et al., PeptideRes., 2:171-177, 1989). The crude product was purified on a Waters C18Sep Pak cartridge followed by a Delta Pak C18 reverse phase column on aWaters model 510 HPLC controlled with a Millenium 2010 chromatographymanager system (Waters, Milford, Mass.) with detection at 214 nm atambient temperature. Solvent A=0.1% aqueous trifluoroacetic acid (TFA);solvent B=80% aqueous acetonitrile containing 0.1% TFA. Conditions:Initial solvent consisting of 20% B was followed by Waters linearprogram 6-100% B over 40 min; flow rate 2 ml/min. Retention timeHca-Asn-Phe-Cpa-Leu-NH₂): 15.0 min. The pure pseudopeptide analog wasanalyzed and quantified by amino acid analysis under previouslydescribed conditions, revealing the following analysis: F(1.0), L(1.0)and N(0.9). A fast atom bombardment (FAB) mass spectrum was obtained byadding 10 g of pseudopeptide sample to glycerol (1.5 μl) on a copperprobe, followed by bombardment with 8 kV Xe atoms on a Kratos MS-50 massspectrometer (Kratos, Manchester, UK). The structural identity and ameasure of the purity of the pseudopeptide was confirmed by the presenceof the following molecular ion (MH⁺): Hca-Asn-Phe-Cpa-Leu-NH₂, 607.4(Calc. MH⁺: 607.32).

[0063] Animals

[0064]D. punctata females mate almost immediately following emergence.Therefore, newly emerged, mated females were transferred each day fromthe stock culture to an incubator and kept at 27±1° C. The relativehumidity was approximately 50% with a 12 hour light:dark cycle. Insectswere reared on Purina Lab Chow and water. Mating was confirmed by thepresence of a spermatophore. Basal oocyte length was also measured (day5=1.44-1.68 mm) only day 5 mated females were utilized for thedegradation studies. Day 7 mated females (only with oviposited basaloocytes) were the source of corpora allata for analysis of JHbiosynthesis.

[0065] Solid Phase Extraction

[0066] Preparation

[0067] Pasteur pipettes were used to make C₁₈ reversed-phase columns. Aglass bead was placed in the bottom of each Pasteur pipette-andapproximately 200 mg 125 Å C₁₈ bulk packing material was added. Glasswool was placed over the-top of the packing material to hold it inplace.

[0068] Procedure

[0069] Each column was washed with 1.5 ml of 0.1% BSA in 0.1% aqueousTFA, followed by 1.5 ml of 40% acetonitrile in 0.1% TFA and finally, 1.5ml of 0.1% aqueous TFA. Samples were diluted in 0.2 ml 0.1% aqueous TFAand applied to column. Eluant was reapplied to the column 2×. The columnwas washed with 1.5 ml 0.1% aqueous TFA followed by 17% acetonitrile in0.1% aqueous TFA. The allatostatins and their metabolites were elutedwith 0.5 ml 40% acetonitrile in 0.1% aqueous TFA into 12 mm X 75 mmculture tubes and were dried with a Speed-Vac.

[0070] Protein Assay

[0071] Protein content of membrane preparations was determined by themethod of Bradford (Bradford, Anal. Biochem., 72:248-254, 1976) usingBSA as standard. The average of three separate determinations was used.

[0072] Allatostatin Degradation Assay

[0073] Tissue Collection

[0074] Day 5 mated female D. punctata were anaesthetized on ice beforedissection. Following dissection, tissues were stored in saline. (0.9%NaCl pH 7.0-7.2 or cockroach saline; NaCl 150 μM, KCl 12 μM, CaCl₂.6H₂O10 μM MgCl₂. 6H₂O 3 μM, glucose 40 μM, HEPES 10 μM at pH 7.2-7.4) onice. Tissues were cleaned of fat body and trachea and guts were cleaned.Midgut peritrophic membrane was removed, midguts cut open, the gutsgently pulled through forceps and then washed 3×. Tissues were stored at−70° C. until sufficient material was collected.

[0075] Membrane preparation

[0076] Tissues were pooled and homogenized in saline on ice inmicrocentrifuge tubes (2.5 ml) for 2 min with an Omni hand heldhomogenizer. Homogenates were centrifuged at 1000 g for ten min at 4° C.to remove cellular debris. The pellet was discarded. Supernatant wassubsequently centrifuged at 30,000 g for 30 min. Pellet (crude membranepreparation) was washed three times in homogenization buffer andresuspended in saline using the Omni homogenizer.

[0077] Hemolymph Preparation

[0078] Hemolymph was collected following the method of King and To be(Insect Biochem., 0.18:793-805, 1988). Hemolymph was diluted 100× withsaline.

[0079] Assay

[0080] Crude membrane preparation or hemolymph was aliquoted into 1.5 mlmicrocentrifuge tubes for in vitro assay. Dip-AST5 (6 μM) or analog wasadded to each assay tube from a 1 mM stock solution. Total incubationvolume was 500 μl. Tubes were placed on a shaker at room temp for theduration of the incubation time. Incubations were terminated by theaddition of 200 μl 30% aqueous TFA. Samples were centrifuged at 10,000 gand the supernatant was applied to a C₁₈ solid phase extraction column.None of the potential, synthetic Dip-AST5 metabolites are lost(unpublished data). The 17-40% acetonitrile fraction was concentratedwith a Speed-Vac apparatus and held at −70° C. until HPLC analysis.Samples were diluted in 0.5 ml 0.1% aqueous TFA, filtered through 0.2 μmmicro-spin filters and injected onto the HPLC column. Separation ofallatostatins and their catabolites, or analogs and their catabolites,was performed using reversed-phase HPLC (see HPLC methods). Whenavailable, retention times of synthetic metabolites were used as areference to identify endogenous metabolites. Dip-AST5 fragments weresynthesized by the Institute for Bibsciences and Technology, Dept. ofEntomology, Texas A & M University. Dip-ASTs were synthesized by theCore. Facility of Insect Biotech Canada (Department of Biochemistry,Queen's University) or obtained from Sigma Chemical Co., St. Louis, Mo.63178 USA. The identity of fragments resulting from peptidasedegradation was also confirmed by FAB mass spectrometry by addingsamples in a glycerol medium (1.5 μl) onto a copper probe, followed bybombardment with 8 kV Xe atoms on a Kratos MS-50 mass spectrometer.(Kratos, Manchester, UK).

[0081] HPLC

[0082] Degradation experiments were analyzed by RP-HPLC using a 220×4.6mm Brownlee Phenyl column (5 μm) on a Spectra-Physics chromatographysystem with a Spectra-Physics Chromjet integrator and a Spectra-Physics8490 detector. Following a 5 min wash with 1.5% acetonitrile in 0.1%aqueous TFA, a linear gradient of acetonitrile (15-37.5% in 50 min;37.5-65% in 5 min) at a flow rate of 0.5 ml/min was used to elutepeptides and analogs. Fractions (0.5 ml) were collected each minuteduring the gradient run.

[0083] Radiochemical Assay

[0084] Radiochemical assays were conducted as described in Example 1.

[0085] Controls

[0086] Controls were prepared and conducted as described in Example 1.

Results

[0087] In analog 396-1 (Ala-Arg-Pro-Tyr-Asn-Aic-Gly-Leu-NH₂) the Pheresidue was replaced with an indane ring system (abbreviatedAic=2-Amino-indane-2-carboxyl-; see FIG. 1) which preserved the presenceof the side chain phenyl ring, a structural feature critical forbiological activity (Hayes et al., ibid). In a second analog (397-2:Ala-Arg-Pro-Tyr-Asn-Phe-Cpa-Leu-NH₂), the-Gly residue was replaced witha cyclopropyl ring system (abbreviated Cpa=cyclopropylAla-, see FIG. 2).Molecular dynamics analyses, incorporating distance and angleconstraints obtained from NMR spectroscopy, were conducted separatelyfor each of the analogs of the series. All demonstrated a preference fora very similar low energy turn over the four C-terminal residues.

[0088] Both allatostatin analogs 396-1 and 397-2 retained significantbioactivity (Table 2); specifically, 396-1 has an IC₅₀=2.12 nM whichcompares favorably to Dip-AST5 which has an IC₅₀=3.1 n Patterns ofpeptidase hydrolysis by hemolymph enzymes for analogs 396-1 and 397-2indicate that the analogs were targeted at peptide bonds outside of theactive core region. The analogs were cleaved near the N-terminus,initially at Arg-Pro, followed by cleavage at Pro-Tyr. These sites ofcleavage are directly comparable to the sites of cleavage in the naturalpeptide Dip-AST5 by soluble hemolymph enzymes (Arg-Leu and Leu-Tyr)(Garside et al., Peptides, 18:17-25, 1997).

[0089] Surprisingly, these analogs showed little resistance todegradation by enzymes in crude membrane preparations. Whereas themodifications successfully prevented truncation of the C-terminalLeu-NH₂, both analogs were still degraded at secondary cleavage sitesnear the N-terminus, similar to those observed following incubation ofallatostatins with hemolymph enzymes. Membrane-bound (brain and midgut)peptidases cleaved 396-1 first at Ala-Arg, followed by cleavage atPro-Tyr-and then at Asn-Aic yielding the C-terminal tripeptide(Aic-Gly-Leu-NH₂). Analog 397-2 was initially cleaved at Arg-Profollowed by cleavage at Tyr-Asn. Interestingly, we found no evidence ofaminopeptidase removal of the N-terminal Ala in analog 0.397-2.

[0090] Since cleavage of analogs 396-1 and 397-2 occurred near theN-terminus, we synthesized analog AST(b)φ2 (FIG. 1), which retained thecyclopropyl ring system of 397-2 at the C-terminus and also incorporatedan unnatural, non-amino acid group, hydrocinnamic acid, at theN-terminus. The hydrocinnamic acid group replaces the criticalN-terminal Tyr, thereby effectively removing both cleavage sitessuggested from the incubation of Dip-ASTs with hemolymph enzymes. Thisalso effectively removed the sites targeted by membrane-bound enzymes inanalog 397-2. The hydrocinnamic acid moiety lacks the phenolic OH groupand N-terminal amino group of the Tyr residue. This analog exhibitedextreme resistance to degradation by enzymes in hemolymph and in crudemembrane preparations of brain and midgut (Table 1). Assay of AST(b)φ2for inhibition of JH biosynthesis indicates that it does retainsignificant biological activity (FIG. 4).

Discussion

[0091] Studies on the structure-activity relationships of theallatostatins indicate that the C-terminal pentapeptideTyr/Phe-Xaa-Phe-Gly-Leu/Ile-NH₂ represents the core sequence requiredfor functional allatostatin activity in vitro (Duve et al., Proc. Natl.Acad. Sci. USA, 90:2456-2460, 1993; Hayes et al. ibid; Pratt et al.,:1989, ibid; Pratt et al., 1991, ibid). In terms of the inhibition of JHbiosynthesis, the amino acid side chain of Leu was the most importantside chain, followed by Phe and Tyr, all of which are located in theC-terminal core sequence. Recent studies have elucidated the primarycatabolic cleavage sites of allatostatins following incubation witheither soluble-enzymes in the hemolymph or with membrane peptidasesin-crude membrane preparations (Garside et al., Peptides, 18:17-25,1997; and Garside et al., Gen. Comp. Endocrinol., 108:258-270, 1997).Hemolymph enzymes cleave Dip-AST5 primarily near the N-terminus atAr-Leu, to yield the C-terminal hexapeptide. This hexapeptide issubsequently cleaved at Leu-Tyr to yield the C-terminal pentapeptide(Garside et al., Peptides, 0.18:17-25, 1997). Interestingly, thesecleavages do not inactivate Dip-AST5 because they do not target siteswithin the core sequence of the allatostatins. However, the potency ofthese two catabolites is significantly reduced (Stay et al.,Allatostatins, neuropeptide inhibitors of juvenile hormone biosynthesisin brain and corpora allata of the cockroach Dipioptera punctata, In:Menn et al., Eds., Insect Neuropeptides, Washington D.C., AmericanChemical Society, 164-176, 1991). For example, the activity of theC-terminal pentapeptide is reduced about 1000-fold (Bendena et al.,ibid). Catabolic enzymes in crude membrane preparations of brain, gutand corpora allata cleave allatostatins primarily in the core C-terminalregion at Gly-Leu-NH₂, with a secondary cleavage site between Tyr-Xaa.Cleavage at the primary site completely inactivates the allatostatin interms of the inhibition of JH biosynthesis.

[0092] Analogs 396-1 and 397-2 were constructed with modifications inthe C-terminal ‘active core’ region of the allatostatins to blockdegradative cleavage by membrane-bound enzymes. Surprisingly, theseanalogs showed little resistance to cleavage by membrane-bound enzymes.Nevertheless, the two analogs did block the membrane-bound degradationthat would occur at the primary cleavage site within the C-terminalactive core pentapeptide region, between Gly-Leu-NH₂ in native peptide.The observed cleavage pattern of 396-1 by membrane-bound (brain andmidgut) peptidases is consistent with aminopeptidase removal of Alafollowed by two consecutive dipeptidyl amingpeptidase-like (DAP)cleavages. In analog 397-2, the two observed cleavages are alsoconsistent with DAP-like removal of successive N-terminal dipeptides.Alternatively, the second cleavage observed-with 397-2 (Tyr-Asn) may becatalyzed by a chymotrypsin-like enzyme, because it occurs on thecarboxyl side of an aromatic residue.

[0093] This N-terminal cleavage pattern observed with analogs 396-1 and397-2 provided the impetus for the design of analog AST(b)φ2,incorporating the cyclopropyl ring system from 397-2 and a hydrocinnamicacid group, a mimic of the Tyr residue, to ‘cap’ the N-terminus. Thisanalog effectively removed cleavage sites suggested from incubation ofDip-ASTS with hemolymph enzymes, and those sites targeted bymembrane-bound enzymes in native allatostatins and in analog 397-2. Theobserved results indicate that AST(b)φ2 is the first mimetic analog ofan insect neuropeptide with resistance to both hemolymph andtissue-bound peptidases. This analog demonstrated 8-fold and 600-foldlonger half-lives than the native neuropeptide Dip-AST5 followingexposure to hemolymph and brain peptidases, respectively, and proved tobe completely resistant to midgut peptidases.

[0094] It is understood that the foregoing detailed description is givenmerely by way of illustration and that modifications and variations maybe made therein without departing from the spirit and scope of theinvention. TABLE 1 Half-life of Dip-AST5 and AST-analogs Haemolymph¹Brain² Midgut² AST/AST-analog (min) (min) (min) Dip-AST5  153.0 18.367.9 396-1 ND 17.2 22.6 397-2 ND 60.9 20.8 AST(b)φ2 1214.7 10771.5 ∞

[0095] TABLE 2 in vitro inhibition of JH biosynthesis by Dip-AST5 andAST analogs AST/AST-analog IC₅₀ ¹ Dip-AST5 3.12 × 10⁻⁹ M 396-1 2.12 ×10⁻⁹ M 397-2 1.72 × 10⁻⁷ M AST(b)φ2 1.55 × 10⁻⁶ M

[0096]

1 14 1 13 PRT Diploptera punctata 1 Ala Tyr Ser Tyr Val Ser Glu Tyr LysArg Leu Pro Val 1 5 10 2 4 PRT Diploptera punctata 2 Asp Gly Arg Met 1 38 PRT Diploptera punctata 3 Ala Pro Ser Gly Ala Gln Arg Leu 1 5 4 4 PRTDiploptera punctata 4 Gly Gly Ser Leu 1 5 5 PRT Diploptera punctata 5Gly Asp Gly Arg Leu 1 5 6 11 PRT Diploptera punctata 6 Pro Val Asn SerGly Arg Ser Ser Gly Ser Arg 1 5 10 7 6 PRT Diploptera punctata 7 Tyr ProGln Glu His Arg 1 5 8 14 PRT Diploptera punctata VARIANT (14) May be Tyror Phe 8 Ala Tyr Ser Tyr Val Ser Glu Tyr Lys Arg Leu Pro Val Xaa 1 5 109 5 PRT Diploptera punctata VARIANT (5) May be Tyr or Phe 9 Asp Gly ArgMet Xaa 1 5 10 9 PRT Diploptera punctata VARIANT (9) May be Tyr or Phe10 Ala Pro Ser Gly Ala Gln Arg Leu Xaa 1 5 11 5 PRT Diploptera punctataVARIANT (5) May be Tyr or Phe 11 Gly Gly Ser Leu Xaa 1 5 12 6 PRTDiploptera punctata VARIANT (6) May be Tyr or Phe 12 Gly Asp Gly Arg LeuXaa 1 5 13 12 PRT Diploptera punctata VARIANT (12) May be Tyr or Phe 13Pro Val Asn Ser Gly Arg Ser Ser Gly Ser Arg Xaa 1 5 10 14 7 PRTDiploptera punctata VARIANT (7) May be Tyr or Phe 14 Tyr Pro Gln Glu HisArg Xaa 1 5

We claim:
 1. A compound of the formula R—X₁-Phe-Gly-X₂—NH₂ wherein X₁ isselected from the group consisting of Asn, Asp, Gly, Ser, and Ala, andX₂ is selected from the group consisting of Leu and Ile, and R isselected from the group consisting of: Cb-(CH₂)_(n)—C(O)—X_(3′—)  (a)wherein (I) Cb is a carborane, (ii) n is 1, 2, or 3, and (iii) X₃′ isselected from the group consisting of a bond, Tyr, Phe, and carboranylalanine, said carboranyl alanine having the structure:

R₁L_(m)-X₄—R₂—X₃—  (b) wherein (I) X₄ is selected from the groupconsisting of a bond and Arg, (ii) R₁ is a hydrophobic moiety selectedfrom the group consisting of aromatic containing amine groups, aromaticcontaining acyl groups, and aliphatic fatty acyl groups, saidhydrophobic moiety being effective to render said compound amphiphilic,(ii) m is 0 or 1, (iii) L is a spacer which, when R₁ is an aromaticcontaining acyl group or aliphatic fatty acyl group, said spacer isselected from the group consisting of non-polar hydrocarbon groupshaving an amino group and an acyl group, and uncharged α-amino acids, orwhen R₁ is an aromatic containing amine group, said spacer is a diacylgroup, (iv) R₂ is selected from the group consisting of a bond, an aminoacid, and a polypeptide group, said polypeptide group comprising all ora portion of an allatostatin neuropeptide which is naturally contiguousto the C terminal pentapeptide X₃—X₁-Phe-Gly-X₂—NH₂, and which saidpolypeptide group is sufficiently small as to retain the hydrophobicityof said compound introduced by said hydrophobic moiety, and (v) X₃ isselected from the group consisting of Tyr, Phe, and carboranyl alanine;and (c) R₃-carboranyl alanine-, wherein R₃ is selected from the groupconsisting of H and Arg.
 2. The compound of claim 1 wherein R is saidCb-(CH₂)_(n)—C(O)—X₃′—.
 3. The compound of claim 2 wherein saidcarborane is selected from the group consisting of o-carborane andm-carborane.
 4. The compound of claim 1 wherein R is saidR₁-L_(m)-X₄—R₂—X₃—.
 5. The compound of claim 4 wherein R₂ is a bond. 6.The compound of claim 4 wherein R₂ is selected from the group consistingof: -Leu-, -Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-  Val-,-Ser-Lys-Met-, -Asp-Gly-Arg-Met-, -Asp-Arg-Leu-, -Ala-Arg-Pro-,-Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-, -Gly-Gly-Ser-Leu-,-Gly-Asp-Gly-Arg-Leu-, -Pro-Val-Asn-Ser-Gly-Arg-Ser-Ser-Gly-Ser-Arg-,-Tyr-Pro-Gln-Glu-His-Arg-, and -Pro-.


7. The compound of claim 4 wherein R₁ is a hydrophobic aromaticcontaining acyl group, m is 1, and L is selected from the groupconsisting of non-polar hydrocarbon groups having a free amino group andfree acyl group, and uncharged α-amino acids.
 8. The compound of claim 7wherein R₁ is selected from the group consisting of phenyl alkanoic acylgroups, phenyl alkenoic acyl groups, and phenyl alkynoic acyl groups. 9.The compound of claim 8 wherein R₁ is selected from the group consistingof a 9-fluoreneacetic acid group, a 6-phenyl hexanoic acyl group; and a9-phenyl nonanoic acyl group.
 10. The compound of claim 7 wherein L isselected from the group consisting of Ala, Ala-Ala, and Gly.
 11. Thecompound of claim 4 wherein R₁ is a hydrophobic aromatic acid and m is0.
 12. The compound of claim 11 wherein R₁ is selected from the groupconsisting of phenyl alkanoic acyl groups, phenyl alkenoic acyl groups,and phenyl alkynoic acyl groups.
 13. The compound of claim 12 wherein R₁is selected from the group consisting of a 9-fluoreneacetic acid group,a 6-phenyl hexanoic acyl group, and a 9-phenyl nonanoic acyl group. 14.A composition comprising the compound of claim 1 and an inert carrier.15. The composition of claim 14 wherein said carrier is water.
 16. Amethod for controlling insects comprising applying the compound of claim1 to the locus of said insects.
 17. The method of claim 16 wherein saidinsects are cockroaches.
 18. The method of claim 16 wherein saidapplying comprises topically applying said compound onto said insects.19. The method of claim 17 wherein said compound is applied in an amounteffective to inhibit juvenile hormone production by said insect.
 20. Acompound of the formula: R′—X₁—X_(a)—X_(b)—X₂—NH₂ wherein: (a) R′ isselected from the group consisting of Phe, Tyr, a hydrocinnamyl group, ap-hydroxyhydrocinnamic acyl group, and a polypeptide group, which saidpolypeptide group comprises all or a portion of an allatostatinneuropeptide which is naturally contiguous to the C terminaltetrapeptide X₁-Phe-Gly-X₂—NH₂; (b) X₁ is selected from the groupconsisting of Asn, Asp. Gly, Ser, and Ala; (C) X₂ is selected from thegroup consisting of Leu and Ile; (d) X_(a)—X_(b) is selected from thegroup consisting of: (I) -Phe-Caa- wherein Caa is a cycloalkyl alaninegroup selected from the group consisting of cylcopropyl alanine havingthe structure:

 or cyclobutyl alanine having the structure:

(ii)-Aic-Gly- wherein Aic is a 2-amino-indane-2-carboxyl group havingthe structure:

(iii)-Bzd- wherein Bzd is a 1,4-benzodiazepine group having thestructure:

and (iv) -Aic-Caa-.
 21. The compound of claim 20 wherein R′ is Phe orTyr.
 22. The compound of claim 20 wherein R′ is a hydrocinnamyl group ora hydroxycinnamyl group.
 23. The compound of-claim 20 wherein R₁ isselected from the group consisting of: Leu-X₃-,Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro- Val-X₃-,Ser-Lys-Met-X₃-, Asp-Gly-Arg-Met-X₃-, Asp-Arg-Leu-X₃-, Ala-Arg-Pro-X₃-,Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-X₃-, Gly-Gly-Ser-Leu-X₃-,Gly-Asp-Gly-Arg-Leu-X₃-,Pro-Val-Asn-Ser-Gly-Arg-Ser-Ser-Gly-Ser-Arg-X₃-,Tyr-Pro-Gln-Glu-His-Arg-X₃-, and Pro-X₃-

wherein X₃ is Tyr or Phe.
 24. The compound of claim 20 whereinX_(a)-X_(b) is said Phe-cycloalkyl alanine.
 25. The-compound of claim 22wherein X_(a)-X_(b) is said Phe-cycloalkyl alanine.
 26. The compound ofclaim 20 wherein X_(a)-X_(b) is said Aic-Gly.
 27. The compound of claim22 wherein X_(a)-X_(b) is said Aic-Gly.
 28. The compound of claim 20wherein X_(a)-X_(b) is said Aic-Caa.
 29. The compound of claim 22wherein X_(a)-X_(b) is said Aic-Caa.
 30. The compound of claim 20wherein X_(a)-X_(b) is said Bzd.
 31. The compound of claim 22 whereinX_(a)-X_(b) is said Bzd.
 32. A composition comprising the compound ofclaim 20 and an inert carrier.
 33. The composition of claim 32 whereinsaid carrier is water.
 34. A method for controlling insects comprisingapplying the compound of claim 20 to the locus of said insects.
 35. Themethod of claim 34 wherein said insects are cockroaches.
 36. The methodof claim 34 wherein said compound is applied in an amount effective toinhibit juvenile hormone production by said insect.
 37. A method forcontrolling insects comprising applying the compound of claim 30 to thelocus of said insects in an amount effective to stimulate juvenilehormone production by said insect.
 38. A compound of the formula:R—X₁—X_(a)—X_(b)—X₂—NH₂ wherein X₁ is selected from the group consistingof Asn, Asp, Gly, Ser, and Ala; X₂ is selected from the group consistingof Leu and Ile; R is selected from the group consisting of:Cb-(CH₂)_(n)—C(O)—X_(3′—)  (a) wherein (I) Cb is a carborane, (ii) n is1, 2, or 3, and (iii) X₃ ¹ is selected from the group consisting of abond, Tyr, Phe, and carboranyl alanine, said carboranyl alanine havingthe structure:

R₁-L_(m)-X₄—R₂—X₃—  (b) wherein (I) X₄ is selected from the groupconsisting of a bond and Arg, (ii) R₁ is a hydrophobic moiety selectedfrom the group consisting of aromatic containing amine groups, aromaticcontaining acyl groups, and aliphatic fatty acyl groups, saidhydrophobic moiety being effective to render said compound amphiphilic,(ii) m is 0 or 1, (iii) L is, a spacer which, when R₁ is an aromaticcontaining acyl group or an aliphatic fatty acyl group, said spacer isselected from the group consisting of non-polar hydrocarbon groupshaving an amino group and an acyl group, and uncharged α-amino acids, orwhen R₁ is an aromatic containing amine group, said spacer is a diacylgroup, (iv) R₂ is selected from the group consisting of a bond, an aminoacid, and a polypeptide group, said polypeptide group comprising all ora portion of an allatostatin neuropeptide which is naturally contiguousto the C terminal pentapeptide X₃—X₁-Phe-Gly-X₂—NH₂, and which saidpolypeptide group is sufficiently small as to retain the hydrophobicityof said compound introduced by said hydrophobic moiety, and (v) X₃ isselected from the group consisting of Tyr, Phe, and carboranyl alanine;and (c) R₃-carboranyl alanine-, wherein R₃ is selected from the groupconsisting of H and Arg; and X_(a)-X_(b) is selected from the groupconsisting of: (a) -Phe-Caa- wherein Caa is a cycloalkyl alanine groupselected from the group consisting of cylcopropyl alanine having-thestructure:

 or cyclobutyl alanine having the structure:

(b)-Aic-Gly- wherein Aic is a 2-amino-indane-2-carboxyl group having thestructure:

(c)-Bzd- wherein Bzd is a 1,4-benzodiazepine group having the structure:

 and (d) -Aic-Caa-.
 39. A composition comprising the compound of claim38 and an inert carrier.
 40. A method for controlling insects comprisingapplying the compound of claim 38 to the locus of said insects.